Abstract
In this study, we aimed to study the role of miRNAs in intrauterine adhesion (IUA) disease. An IUA cell model was constructed by TGF-β1. Smad3 inhibitor (SIS3) can inhibit the Smad3 signaling pathway and affect the role of TGF-β1; thus, it was used to identify the role of Smad3 and related miRNAs in IUA. Cell number significantly increased in the TGF-β1 group after 72 h and 96 h, respectively, compared with that in the control group (P < 0.05). However, cell proliferation was significantly decreased in the TGF-β1 + SIS3 group (P < 0.0001). Cell apoptosis was increased in the TGF-β1 + SIS3 group compared with that in the TGF-β1 group. Western Blot (WB) analysis suggested that TGF-β1 treatment could effectively increase the expression of α-SMA, COL1, Smad3, and p-Smad3, which could be inhibited by SIS3 treatment. A total of 235 and 530 differentially expressed miRNAs in the TGF-β1 + SIS3 group were significantly up- and downregulated compared with those in the TGF-β1 group, respectively. These differentially expressed miRNAs were enriched in the MAPK and PI3K-AKT pathways. The ten most differentially expressed miRNAs were selected to verify their expressions using quantitative real-time polymerase chain reaction (qPCR). Furthermore, overexpression of rno-miR-3586-3p and rno-miR-455-5p can promote cell proliferation and exacerbate the IUA pathogenic process. However, overexpression of rno-miR-204-3p and rno-miR-3578 can inhibit cell behavior and IUA progression. The above results can provide detailed information for the understanding of IUA molecular mechanisms.
Similar content being viewed by others
Data availability
Raw sequencing data presented in this paper are available under the GEO accession number PRJNA598792.
References
Deans R, Abbott J (2010) Review of intrauterine adhesions. J Minim Invasive Gynecol 17(5):555–569. https://doi.org/10.1016/j.jmig.2010.04.016
Zhu HY, Ge TX, Pan YB, Zhang SY (2017) Advanced role of hippo signaling in endometrial fibrosis: implications for intrauterine adhesion. Chin Med J 130(22):2732–2737. https://doi.org/10.4103/0366-6999.218013
Conforti A, Alviggi C, Mollo A, De Placido G, Magos A (2013) The management of Asherman syndrome: a review of literature. Reprod Biol Endocrinol 11:118. https://doi.org/10.1186/1477-7827-11-118
Cao Y, Sun H, Zhu H, Zhu X, Tang X, Yan G, Wang J, Bai D, Wang J, Wang L, Zhou Q, Wang H, Dai C, Ding L, Xu B, Zhou Y, Hao J, Dai J, Hu Y (2018) Allogeneic cell therapy using umbilical cord MSCs on collagen scaffolds for patients with recurrent uterine adhesion: a phase I clinical trial. Stem Cell Res Ther 9(1):192. https://doi.org/10.1186/s13287-018-0904-3
March CM (2011) Management of Asherman's syndrome. Reprod Biomed Online 23(1):63–76. https://doi.org/10.1016/j.rbmo.2010.11.018
Bai X, Liu J, Cao S, Wang L (2019) Mechanisms of endometrial fibrosis and the potential application of stem cell therapy. Discov Med 27(150):267–279
Szostek-Mioduchowska AZ, Lukasik K, Skarzynski DJ, Okuda K (2019) Effect of transforming growth factor-beta1 on alpha-smooth muscle actin and collagen expression in equine endometrial fibroblasts. Theriogenology 124:9–17. https://doi.org/10.1016/j.theriogenology.2018.10.005
Meng XM, Nikolic-Paterson DJ, Lan HY (2016) TGF-beta: the master regulator of fibrosis. Nat Rev Nephrol 12(6):325–338. https://doi.org/10.1038/nrneph.2016.48
Nagaraj NS, Datta PK (2010) Targeting the transforming growth factor-beta signaling pathway in human cancer. Expert Opin Investig Drugs 19(1):77–91. https://doi.org/10.1517/13543780903382609
Salma U, Xue M, Ali Sheikh MS, Guan X, Xu B, Zhang A, Huang L, Xu D (2016) Role of transforming growth factor-beta1 and smads signaling pathway in intrauterine adhesion. Mediators Inflamm 2016:4158287. https://doi.org/10.1155/2016/4158287
Lu TX, Rothenberg ME (2018) MicroRNA. J Allergy Clin Immunol 141(4):1202–1207. https://doi.org/10.1016/j.jaci.2017.08.034
O'Brien J, Hayder H, Zayed Y, Peng C (2018) Overview of MicroRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol 9:402. https://doi.org/10.3389/fendo.2018.00402
Hammond SM (2015) An overview of microRNAs. Adv Drug Deliv Rev 87:3–14. https://doi.org/10.1016/j.addr.2015.05.001
Bushati N, Cohen SM (2007) MicroRNA functions. Annu Rev Cell Dev Biol 23:175–205. https://doi.org/10.1146/annurev.cellbio.23.090506.123406
O'Reilly S (2016) MicroRNAs in fibrosis: opportunities and challenges. Arthritis Res Ther 18:11. https://doi.org/10.1186/s13075-016-0929-x
Qin W, Chung AC, Huang XR, Meng XM, Hui DS, Yu CM, Sung JJ, Lan HY (2011) TGF-beta/Smad3 signaling promotes renal fibrosis by inhibiting miR-29. J Am Soc Nephrol 22(8):1462–1474. https://doi.org/10.1681/asn.2010121308
Li J, Cen B, Chen S, He Y (2016) MicroRNA-29b inhibits TGF-beta1-induced fibrosis via regulation of the TGF-beta1/Smad pathway in primary human endometrial stromal cells. Mol Med Rep 13(5):4229–4237. https://doi.org/10.3892/mmr.2016.5062
Zheng Q, Wang XJ (2008) GOEAST: a web-based software toolkit for gene ontology enrichment analysis. Nucleic Acids Res 36:W358–W363. https://doi.org/10.1093/nar/gkn276
Xie C, Mao X, Huang J, Ding Y, Wu J, Dong S, Kong L, Gao G, Li CY, Wei L (2011) KOBAS 2.0: a web server for annotation and identification of enriched pathways and diseases. Nucleic Acids Res 39:W316–W322. https://doi.org/10.1093/nar/gkr483
Wynn TA, Ramalingam TR (2012) Mechanisms of fibrosis: therapeutic translation for fibrotic disease. Nat Med 18(7):1028–1040. https://doi.org/10.1038/nm.2807
Pohlers D, Brenmoehl J, Loffler I, Muller CK, Leipner C, Schultze-Mosgau S, Stallmach A, Kinne RW, Wolf G (2009) TGF-beta and fibrosis in different organs: molecular pathway imprints. Biochim Biophys Acta 1792(8):746–756. https://doi.org/10.1016/j.bbadis.2009.06.004
Biernacka A, Dobaczewski M, Frangogiannis NG (2011) TGF-beta signaling in fibrosis. Growth Factors 29(5):196–202. https://doi.org/10.3109/08977194.2011.595714
Wynn TA (2008) Cellular and molecular mechanisms of fibrosis. J Pathol 214(2):199–210. https://doi.org/10.1002/path.2277
Zi Z, Chapnick DA, Liu X (2012) Dynamics of TGF-beta/Smad signaling. FEBS Lett 586(14):1921–1928. https://doi.org/10.1016/j.febslet.2012.03.063
Flanders KC (2004) Smad3 as a mediator of the fibrotic response. Int J Exp Pathol 85(2):47–64. https://doi.org/10.1111/j.0959-9673.2004.00377.x
Derynck R, Zhang YE (2003) Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 425(6958):577–584. https://doi.org/10.1038/nature02006
Wang X, Gao JL, Zhao MM, Zhu HX, Tian YX, Li R, Jiang XH, Yu L, Tian JR, Cui JZ (2018) Therapeutic effects of conditioned medium from bone marrow-derived mesenchymal stem cells on epithelial-mesenchymal transition in A549 cells. Int J Mol Med 41(2):659–668. https://doi.org/10.3892/ijmm.2017.3284
Sakairi T, Hiromura K, Takahashi S, Hamatani H, Takeuchi S, Tomioka M, Maeshima A, Kuroiwa T, Nojima Y (2011) Effects of proteasome inhibitors on rat renal fibrosis in vitro and in vivo. Nephrology 16(1):76–86. https://doi.org/10.1111/j.1440-1797.2010.01367.x
Hu Z-C, Shi F, Liu P, Zhang J, Guo D, Cao X-L, Chen C-F, Qu S-Q, Zhu J-Y, Tang B (2017) TIEG1 represses Smad7-mediated activation of TGF-β1/Smad signaling in keloid pathogenesis. J Investig Dermatol 137(5):1051–1059. https://doi.org/10.1016/j.jid.2016.12.019
Han L, Zhu B, Chen H, Jin Y, Liu J, Wang W (2019) Proteasome inhibitor MG132 inhibits the process of renal interstitial fibrosis. Exp Ther Med 17(4):2953–2962. https://doi.org/10.3892/etm.2019.7329
Xu F, Liu C, Zhou D, Zhang L (2016) TGF-beta/SMAD pathway and its regulation in hepatic fibrosis. J Histochem Cytochem 64(3):157–167. https://doi.org/10.1369/0022155415627681
Khalil H, Kanisicak O, Prasad V, Correll RN, Fu X, Schips T, Vagnozzi RJ, Liu R, Huynh T, Lee SJ, Karch J, Molkentin JD (2017) Fibroblast-specific TGF-beta-Smad2/3 signaling underlies cardiac fibrosis. J Clin Invest 127(10):3770–3783. https://doi.org/10.1172/jci94753
Gebert LFR, MacRae IJ (2019) Regulation of microRNA function in animals. Nat Rev Mol Cell Biol 20(1):21–37. https://doi.org/10.1038/s41580-018-0045-7
Rutnam ZJ, Wight TN, Yang BB (2013) miRNAs regulate expression and function of extracellular matrix molecules. Matrix Biol 32(2):74–85. https://doi.org/10.1016/j.matbio.2012.11.003
Liu X, Duan H, Zhang HH, Gan L, Xu Q (2016) Integrated data set of microRNAs and mRNAs involved in severe intrauterine adhesion. Reprod Sci 23(10):1340–1347. https://doi.org/10.1177/1933719116638177
Zhang Y, Huang XR, Wei LH, Chung AC, Yu CM, Lan HY (2014) miR-29b as a therapeutic agent for angiotensin II-induced cardiac fibrosis by targeting TGF-beta/Smad3 signaling. Mol Ther 22(5):974–985. https://doi.org/10.1038/mt.2014.25
Ramdas V, McBride M, Denby L, Baker AH (2013) Canonical transforming growth factor-beta signaling regulates disintegrin metalloprotease expression in experimental renal fibrosis via miR-29. Am J Pathol 183(6):1885–1896. https://doi.org/10.1016/j.ajpath.2013.08.027
Roderburg C, Urban GW, Bettermann K, Vucur M, Zimmermann H, Schmidt S, Janssen J, Koppe C, Knolle P, Castoldi M, Tacke F, Trautwein C, Luedde T (2011) Micro-RNA profiling reveals a role for miR-29 in human and murine liver fibrosis. Hepatology 53(1):209–218. https://doi.org/10.1002/hep.23922
Suzuki HI (2018) MicroRNA control of TGF-beta signaling. Int J Mol Sci. https://doi.org/10.3390/ijms19071901
Cai C, He H, Duan X, Wu W, Mai Z, Zhang T, Fan J, Deng T, Zhong W, Liu Y, Zhong W, Zeng G (2018) miR-195 inhibits cell proliferation and angiogenesis in human prostate cancer by downregulating PRR11 expression. Oncol Rep 39(4):1658–1670. https://doi.org/10.3892/or.2018.6240
Zhang YX, Yan YF, Liu YM, Li YJ, Zhang HH, Pang M, Hu JX, Zhao W, Xie N, Zhou L, Wang PY, Xie SY (2016) Smad3-related miRNAs regulated oncogenic TRIB2 promoter activity to effectively suppress lung adenocarcinoma growth. Cell Death Dis 7(12):e2528. https://doi.org/10.1038/cddis.2016.432
Martinez-Anton A, Sokolowska M, Kern S, Davis AS, Alsaaty S, Taubenberger JK, Sun J, Cai R, Danner RL, Eberlein M, Logun C, Shelhamer JH (2013) Changes in microRNA and mRNA expression with differentiation of human bronchial epithelial cells. Am J Respir Cell Mol Biol 49(3):384–395. https://doi.org/10.1165/rcmb.2012-0368OC
Chai J, Wang S, Han D, Dong W, Xie C, Guo H (2015) MicroRNA-455 inhibits proliferation and invasion of colorectal cancer by targeting RAF proto-oncogene serine/threonine-protein kinase. Tumour Biol 36(2):1313–1321. https://doi.org/10.1007/s13277-014-2766-3
Mikhaylova O, Stratton Y, Hall D, Kellner E, Ehmer B, Drew AF, Gallo CA, Plas DR, Biesiada J, Meller J, Czyzyk-Krzeska MF (2012) VHL-regulated MiR-204 suppresses tumor growth through inhibition of LC3B-mediated autophagy in renal clear cell carcinoma. Cancer Cell 21(4):532–546. https://doi.org/10.1016/j.ccr.2012.02.019
Salinas-Vera YM, Marchat LA, García-Vázquez R, González de la Rosa CH, Castañeda-Saucedo E, Tito NN, Flores CP, Pérez-Plasencia C, Cruz-Colin JL, Carlos-Reyes Á, López-González JS, Álvarez-Sánchez ME, López-Camarillo C (2018) Cooperative multi-targeting of signaling networks by angiomiR-204 inhibits vasculogenic mimicry in breast cancer cells. Cancer Lett 432:17–27. https://doi.org/10.1016/j.canlet.2018.06.003
Funding
This study was supported by the National Natural Science Foundation of China (Grant No. 81701395).
Author information
Authors and Affiliations
Contributions
YX conceived and designed the study, and critically revised the manuscript. SL performed the experiments, analyzed the data and drafted the manuscript. XH, YL and DS participated in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflicts of interest
The authors declare that they have no conflict of interest.
Consent for publication
All authors have read the manuscript and authorized the submission for publication.
Ethical approval
All procedures performed in studies involving animals were in accordance with the ethical standards of the Experimental Animal Welfare Ethics Branch of Biomedical Ethics Committee of Peking University [permit no. LA2018022]. Appropriate guidelines for the use of animals have been followed.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Liu, S., Huang, X., Liu, Y. et al. Functional analysis of miRNAs combined with TGF-β1/Smad3 inhibitor in an intrauterine rat adhesion cell model. Mol Cell Biochem 470, 15–28 (2020). https://doi.org/10.1007/s11010-020-03741-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11010-020-03741-7